Gene Function, Expression and Regulation in Zebrafish

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications, trials & patents

Abstract

From 9/10/2020 through 9/1/2021 Feldman supported zebrafish research of eleven labs/other customers, conducted independent research and engaged in institutional service. Support of Zebrafish Research Led by Labs & Other Customers, Principal Projects: Porter Lab (NICHD): Genetic Dissection and Creation of Human Disease Models of Sterol Metabolism. In previous years the Core used CRISPR-Cas9 technology to create genetic mutant zebrafish lines for the Porter lab in five genes: dhcr7, npc1, npc2, cln3 and ebp - with roles in various steps of cholesterol metabolism. In 2021 a paper on npc2 phenotypes was published, and two new trainees for the Porter lab were onboarded. Stratakis Lab (NICHD): Function of Zebrafish Orthologs to Human Genes Implicated in Disorders of the Pituitary-Adrenal Axis. In previous years, the Core used CRISPR-Cas9 technology to generate zebrafish carrying loss-of-function mutations in four zebrafish orthologs to human genes implicated by the Stratakis lab in human growth anomalies and eight zebrafish orthologs to human adrenal hyperplasia and Cushing disease-associated genes. In 2021, a manuscript summarizing the characterization of one of these genes, gpr101, was published. The core also helped the Stratakis lab generate a precisely edited zebrafish satb1 mutant line with a nonsynonymous AA substitution that is cognate to a human disease-associated mutation of interest. Kemper lab (NHLBI): Function of zebrafish rca2.1. The Kemper lab is interested in zebrafish rca2.1s function, because it has certain similarities to human CD46 that are not found in the mouse genome. We previously generated a line with a 20 KB deletion of the entire rca2.1 locus and phenotype characterization this past year revealed essential roles in growth and cardiac function. We also generated a second rca2.1 mutant allele a standard small CRISPR indel and preliminary non-complementation analysis indicates it produces the same phenotype. Dr. Brown (Office of Dietary Supplements): Role of minerals in bone health. The NICHD Zebrafish Core In spite of Covid delays preventing Dr. Brown from working on site, we have made progress in preparing for a study that will use defined fish feed from the Nutrition Obesity Research Center of the University of Alabama (Birmingham) to explore how certain minerals influence bone health in the presence or absence of adequate vitamin D, a topic of relevance to human bone health and differences between the role of vitamin D between African Americans and other demographic groups. Specifically, we have obtained the feed and we have done toxicology and nutritional analysis, confirming it has the desired nutritional profile and is not harmful to fish. Independent Research by the NICHD Zebrafish Core Optimizing strategies for CRISPR/Cas9-based homology-directed repair (HDR). The Core previously used CRISPR-Cas9 technology in combination with a customized donor DNA to generate a zebrafish line with an atp7a amino acid (AA) substitution as well as novel restriction enzyme recognition sites, the latter assisting in screening and recovery, and we worked with the NICHD Bioinformatics & Scientific Programming Core to create software for generalizing the design of such donor DNAs for any locus of interest. We deposited a manuscript describing this work on BioRXiv, with plans to forward for peer review in the coming year. We also continued a project comparing precise genome editing methods and efficiencies using high-throughput sequencing. This year we explored strategies for recovery by high-throughput sequencing of progeny or gametes from candidate carriers, gaining extensive knowledge of best practices and successfully recovering a cacna1c allele (Golden lab NIDDK) with a targeted AA-alteration. Data analysis, recovery of additional alleles and manuscript preparation for this project will continue into the next year. Cryopreservation and in vitro fertilization of zebrafish sperm. Over the last year, we have continued to focus on improving quality control measures to ensure viability of cryopreserved zebrafish lines and minimize variability in viability. Central to our strategy is pooling and aliquoting sperm from multiple carriers and doing a test IVF from one aliquot. Institutional Service: ACUC Membership. Feldman has served on the NICHD ACUC since 2015 and continued in this capacity this year, meeting monthly to evaluate and decide upon animal-study proposals, renewals and amendments and ad hoc issues relevant to animal welfare. Virtual Take-Your-Child-to-Work Day. With guidance and editing support from the NICHD Office of Communications and a team from the NIH library, Feldman self-filmed a tutorial discussing zebrafish-based research in general and showing how they are bred and how their offspring are collected and observed.